karst: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - karst

Synonyms - ßHeavy spectrin, ßH

Cytological map position - 63C1--63D3

Function - cytoskeletal protein

Keywords - cytoskeleton, junctions

Symbol - kst

FlyBase ID: FBgn0004167

Genetic map position - 3-[5]

Classification - ßHeavy spectrin

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Deng, H., Wang, W., Yu, J., Zheng, Y., Qing, Y. and Pan, D. (2015). Spectrin regulates Hippo signaling by modulating cortical actomyosin activity. Elife 4: e06567. PubMed ID: 25826608
Summary:
The Hippo pathway controls tissue growth through a core kinase cascade that impinges on the transcription of growth-regulatory genes. Understanding how this pathway is regulated in development remains a major challenge. Recent studies suggested that Hippo signaling can be modulated by cytoskeletal tension through a Rok-myosin II pathway. How cytoskeletal tension is regulated or its relationship to the other known upstream regulators of the Hippo pathway remains poorly defined. This study identifies the spectrins, α-spec, β-spec, or βH-spec contractile proteins at the cytoskeleton-membrane interface, as an upstream regulator of the Hippo signaling pathway. In contrast to canonical upstream regulators such as Crumbs, Kibra, Expanded, and Merlin, spectrin regulates Hippo signaling in a distinct way by modulating cortical actomyosin activity through non-muscle myosin II. These results uncover an essential mediator of Hippo signaling by cytoskeleton tension, providing a new entry point to dissecting how mechanical signals regulate Hippo signaling in living tissues.

BIOLOGICAL OVERVIEW

The gene karst, which codes for the protein ßHeavy-spectrin (ßH) may be confused with, and must be distinguished from the ß-spectrin gene, which codes for the protein ß-spectrin. This overview will first consider the biochemistry and cell biology of karst and then discuss its effects on phenotype. ß spectrins are polymerization partners of alpha Spectrin. an elongated molecule that is a constituent of the submembrane cytoskeleton of epithelial cells. Epithelial cells constitute many tissues of the fly.

The combinatorial association of Drosophila spectra monomers produces either alpha2/ß2 (conventional) or alpha2/ßH2 (Karst-containing) tetramers (Dubreuil, 1990). In this notation the 2's refer to the fact that each tetramer contains two alpha and two beta subunits. Both tetramers crosslink actin and contain ß-chain pleckstein homology (PH) and alpha-chain SH3 domains, supporting the notion that spectrins interact with several cellular components other than actin. Karst, or ßHeavy-spectrin, with 30 spectrin repeats and an Mr = 470 x103, is longer and more massive than conventional ß-spectrins (Mr~250 x103 and 17 repeats). ßHeavy is also unique among ß-isoforms because it (1) contains an SH3 domain and (2) it probably does not bind to ankyrin (Thomas, 1997).

Although ßHeavy-spectrin was first recognized in the fly, it is of ancient origin (Thomas, 1997), has recently been cloned from Caenorhabditis elegans (McKeown, 1998) and may have a vertebrate homolog. In polarized epithelia, alpha-spectrin is generally distributed on plasma membranes; its polymerization partner ß-spectrin is restricted to the basolateral surface, and its second partner, ßH is localized to the apical domain at the adherens junction, on the free apical surface and under brush borders. Recruitment of ß-spectrin to the membrane can occur after adhesion events in fly and vertebrate tissue culture cells. However, during early development in Drosophila, the ßH membrane skeleton becomes polarized prior to formation of mature adherens junctions. This suggests the hypothesis that different ß isoforms may be recruited in different ways (Thomas, 1998 and references).

karst received its name because of the resemblance of the rough eye phenotype to certain karst landforms: domed hills, separated by flat valleys. karst mutant eyes appear roughest towards the posterior margin where ommatidia are sometimes absent. Although the phenotype of the most severe karst mutant eyes resembles that of the EGF receptor mutation DER Ellipse, no dominant interaction has been observed between these loci. karst mutant eyes also exhibit a variable frequency in the percentage of ommatidia that lack photoreceptor R7 (up to 61% exhibit this phenotype). The fact that R7 is the missing photoreceptor has been confirmed by examining serial sections through karst mutant eyes. Ommatidia with only six photoreceptors in distal sections (relative to the brain) have seven photoreceptors in proximal sections. Since R1-6 extend the entire depth of the retina, while R7 extends only through distal regions and R8 replaces R7 in more proximal regions, this result is consistent with the specific absence of R7. karst mutant eyes sometimes (at low frequency) contain ommatidia with very abnormal numbers of photoreceptors. The morphology of such clusters suggests that they may have arisen through fusion of ommatidia to another cluster containing only a small number of photoreceptors. Some photoreceptors have rhabdomeres that are greatly expanded in size. Serial sectioning reveals that these photoreceptors are broader throughout the depth of the retina and do not result from a failure of such cells to extend properly during pupation. The roughening of the eye is 100% penetrant but exhibits variable expressivity. Within ommatidia that do have eight photoreceptors, the arrangement of these cells is not as regular as in wild-type eyes. However, the presence of a recognizable trapezoid in many ommatidia indicates that this patterning event is intact in karst mutants and reveals an equatorial line, so cluster rotation still occurs (Thomas 1998).

To determine if karst belongs in the Sevenless signaling pathway, an epistasis experiment was performed in which the constitutively activated Sevenless receptor, Sev S11 was crossed into a karst mutant background. In a wild-type background, Sev S11 produces an excess of R7 cells because it is expressed in more than one cell per precluster. Siblings with three phenotypes (karst, S11 and S11/karst) were analyzed. If karst is epistatic to Sev S11 (i.e. blocking signaling when penetrant), the mean number of R7s per cluster in S11/karst double mutant flies would be expected to be lower than the mean in S11 flies. Unfortunately, the wider effects of the karst mutation on cell position and rhabdomere morphology prevent a reliable counting of the number of R7-type photoreceptors in S11/karst flies. Nevertheless, the mean number of photoreceptors per ommatidium for the S11/karst flies is not significantly different from the S11 flies. This is consistent with the interpretation that Sev S11 is epistatic to karst, suggesting that karst is upstream of Sevenless or in a parallel pathway contributing to the ligand interaction (Thomas 1998).

It is proposed that the loss of R7 photoreceptors seen in karst mutant eyes is indicative of a reduction in cell adhesion. The commitment of the R7 precursor (pre-R7) to a photoreceptor cell fate requires that the membrane-bound ligand encoded by the bride of sevenless gene (Boss) in R8 must directly contact the Sevenless receptor (SEV) on the surface of pre-R7, and this interaction must sustain a signal for some time to cause this cell fate decision. A subpopulation of Sevenless is seen to concentrate at the adherens junctions, specifically in regions in contact with R8 (Tomlinson, 1987); however, the bulk of both Boss and Sev are more apically restricted (Krämer, 1991; Tomlinson, 1987). If cell adhesion is decreased at the adherens junction, two hypotheses could account for the loss of R7 in karst mutant eyes. (1) A reduction in Boss-Sev encounters might cause signaling to drop below the threshold required to trigger R7 commitment. (2) A pre-R7 or R8 cell might move out of position, preventing contact and thus signaling. In either case, it seems reasonable to expect that the phenotype would be variable as is the case in karst mutant eyes (Thomas 1998).

Two further aspects of the phenotype are consistent with a reduction in cell-cell adhesion: the abberant arrangement of photoreceptors in karst mutant ommatidia and the leakage of hemolymph from abdominal spiracles. With regard to the first condition, enough properly rotated, trapezoidal ommatidia can be identified to indicate that this is probably not a patterning defect. Loosened cell adhesion could readily explain why the cells do not hold their correct relative positions. The second phenotype, a lack of tracheal integrity and hence, hemolymph leakage, might arise because the cells adhere less well to one another. Indeed, it has been observed that dissection to remove the gut of 3rd instar larvae is considerably easier in karst mutant individuals than their heterozygous siblings, because the tracheae are more easily disrupted. Alternatively, the appropriate adhesive contacts may develop incorrectly during pupation leaving gaps in the network (Thomas 1998).

The apical subcellular distribution of ßH/Karst in both the wing and eye imaginal discs colocalizes with Shotgun (DE-cadherin) at the adherens junction, as it does in embryonic epithelia. This association appears intimate: in regions of the eye disc where Shotgun is more abundant, ßH is also more prominent. The mutual exclusivity between ßH and the conventional ß-spectrin isoform is the norm and may be important for cell polarization. While ßH is localized to the adherens junction in imaginal disc epithelia, alpha-spectrin (presumably partnered by ß-spectrin) extends into the basolateral domain. This is consistent with the apical restriction of ßH in other epithelia and the consistent restriction of ß-spectrin to the basolateral membrane (de Cuevas, 1996). To establish such a situation, different proteins must recruit the different spectrins to each domain. In the fly, conventional ß-spectrin is recruited to the membrane by ankyrin (Dubreuil, 1996), while ßH does not colocalize with ankyrin (Lee, 1997) and has no conserved ankyrin-binding site (Thomas, 1997), suggesting that its interaction with the membrane is not ankyrin dependent. Binding to ankyrin could thus be used to specifically recruit ß-spectrin to the basolateral membrane. Presumably the reciprocal situation with some as yet unidentified receptor for ßH results in its apical restriction. This mutually exclusive targeting makes it unlikely that the variable nature of the karst mutation is due to partial redundancy of the two ß-isoforms (Thomas 1998).

The cell adhesion molecule Roughest depends on βHeavy-spectrin during eye morphogenesis in Drosophila

Cell junctions have both structural and morphogenetic roles, and contain complex mixtures of proteins whose interdependencies are still largely unknown. Junctions are also major signaling centers that signify correct integration into a tissue, and modulate cell survival. During Drosophila eye development, the activity of the immunoglobulin cell adhesion molecule Roughest (also known as Irregular chiasm C-roughest protein) mediates interommatidial cell (IOC) reorganization, leading to an apoptotic event that refines the retinal lattice. Roughest and the cadherin-based zonula adherens (ZA) are interdependent and both are modulated by the apical polarity determinant, Crumbs. This study describes a novel relationship between the Crumbs partner βHeavy-spectrin (βH), the ZA and Roughest. Ectopic expression of the C-terminal segment 33 of βH (βH33) induces defects in retinal morphogenesis, resulting the preferential loss of IOC. This effect is associated with ZA disruption and Roughest displacement. In addition, loss-of-function karst and roughest mutations interact to cause a synergistic and catastrophic effect on retinal development. Finally, this study shows that βH coimmunoprecipitates with Roughest and that the distribution of Roughest protein is disrupted in karst mutant tissue. These results suggest that the apical spectrin membrane skeleton helps to coordinate the Cadherin-based ZA with Roughest-based morphogenesis (Lee, 2010).

Investigation of the overexpression of βH segment 33 (βH33) in the eye disc has shown that the phenotype it induces is closely associated with a morphogenetic event that is required for a normal round of apoptosis that refines the retinal lattice. This disruption is correlated with disruption of the ZA and the normal distribution of the Roughest protein, which mediates this morphogenesis. Further experiments demonstrated a strong genetic interaction between loss-of-function karst and rst alleles that appears to result in reduced adhesion between IOCs and ommatidia. A physical association between βH and the Roughest protein was demonstrated by coimmunoprecipitation, and the distribution of Roughest is significantly disrupted in karst mutant cells (Lee, 2010).

As part of a genetic study to probe the function of the βH C-terminal domain (βH33), an overexpression approach was taken to generate a dominant phenotype. It was found that the overexpression of this domain disrupts development, is associated with acridine orange accumulation and can be ameliorated by coexpression of the baculovirus p35 caspase inhibitor, revealing it to be at least in part apoptotic. This is a specific effect of the apical βH isoform, because overexpression of the C-terminus of the related basolateral β-spectrin produces no such phenotype (Lee, 2010).

βH33 contains a PH domain, which is required to induce this phenotype, indicating a requirement for lipid binding or overlapping protein binding. Sequestration of phospholipids is an obvious possible cause of the βH33-induced phenotype and in particular apoptosis. As a class, β-spectrin PH domains appear to use phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] binding as a major mechanism for membrane association. Furthermore, modulation of PtdIns(4,5)P2 levels regulates spectrin association with the Golgi membrane and secretory vesicles. This study demonstrates that the β-spectrin and βH PH domain regions have nearly identical phospholipid specificities, and that they both exhibit the distinct preference for PtdIns(4,5)P2 over PtdIns(3,4,5)P3 seen by Das (2008) for β-spectrin. The only difference is a conspicuous affinity of β-spectrin for phosphatidic acid that is not detectable with βH. This difference probably arises from a second lipid-binding site outside the PH domain, and does not readily account for the lack of phenotype during β-spectrin PH domain expression. The requirement for phospholipid binding in this case might therefore represent only a membrane anchoring mechanism for the ectopic βH33 domain. Finally, these results strongly indicate that bulk differences in phospholipid content are unlikely to be a driving force in the apical-basal polarization of βH and β-spectrin in Drosophila (Lee, 2010).

In the fly eye, a round of apoptosis during pupation eliminates excess IOCs to refine the retinal epithelium. During this period IOCs compete for contact with the primary cells and rearrange into a single row of pigment and bristle cells surrounding each ommatidium, whereas apoptosis culls their numbers to produce an almost crystalline lattice. IOC morphogenesis is mediated by Roughest, which is expressed only in the IOCs at this time where it binds to Hibris expressed on the surface of neighboring primary cells. Roughest colocalizes with the cadherin-based ZA and is both dependent upon the ZA and in turn modulates the properties of the ZA. The results of this study tighten the association between these two adhesion systems, providing a potential common mediator for their activities (Lee, 2010).

During eye development βH is recruited to the membrane by a domain in the Crumbs protein, which specifically regulates ZA stability but not polarity. As a consequence, mutants that lack wild-type βH exhibit a mild and variable disruption of the ZA, but maintain normal apicobasal polarity. This study shows that βH also has a physical association with Roughest during embryonic development; however, it is not yet possible to say whether this is via direct binding or is an indirect association via another protein, as with Crumbs. Physical association with spectrin can result in protein stabilization at the plasma membrane, and so the disruption of Roughest distribution in karst mutant discs strongly suggests that this physical association holds true in the eye. Thus, it is proposed that Crumbs-driven assembly of the apical spectrin-based membrane skeleton provides a means to coordinate the ZA and Roughest/Hibris adhesion systems. If binding of Roughest to βH mediates this coordination at the plasma membrane it must presumably occur when βH is at the ZA before or during cell movement (Lee, 2010).

Emerging data suggest a role for βH in protein trafficking: βH co-isolates with the Golgi-resident protein Lava lamp, and a dramatic reduction in the surface expression level of the apical V-type H+-ATPase in the gut is seen when βH-dependent protein recycling is disrupted in karst mutants. It is therefore possible that βH could play a similar role for Roughest in the eye. The increased levels of Roughest protein in cytoplasmic puncta in karst mutant discs are consistent with this notion. In this context, it is interesting to note that deletion of the Roughest cytoplasmic domain in the rstCT allele, which would be predicted to uncouple it from βH, also leads to elevated levels of vesicular Roughest protein. Because RstCT protein is unlikely to have any residual association with βH, it is suggested that the strong synergism of the rstCT-karst interaction arises from the simultaneous reduction of Roughest and ZA function below a threshold where adhesion is insufficient for IOC-primary cell adhesion (Lee, 2010).

βH33 and karst both disrupt Roughest distribution, but with different consequences: βH33 primarily affects IOC, whereas karst causes selective falling of ommatidia from the retina. It is speculated that the prominent accumulation of Roughest and βH at the IOC-primary cell boundary late in development is necessary to hold the ommatidia in place. Thus, simultaneous reduction in the function of both proteins selectively weakens this interface. By contrast, βH33 would appear to be having its effect earlier during morphogenesis. Given the lack of an obvious direct stimulation of any apoptotic pathway by βH33 expression, coupled with the novel association of βH with Roughest and its known role in cell survival decisions, it is suggested that the apoptotic effects of this domain are an indirect result of the disruption of apicolateral cell junctions that in turn regulate cell survival. It is speculated that the preferential loss of IOCs upon βH33 expression arises because the cell death or survival decisions that are made at this time arise from small differences in cell adhesion in amongst the IOCs that are 'predisposed' to die if a lower threshold is reached: reducing junction function at this time thus greatly, and selectively, increases the number of IOCs that die (Lee, 2010).

Conventional β-spectrins are well known to associate with Ig-CAMs of the L1 subfamily via the adapter molecule Ankyrin. Roughest belongs to a different subfamily that includes Nephrin and has a completely divergent cytoplasmic domain that lacks the conventional Ankryin binding site. Similarly, βH lacks the canonical Ankyrin binding domain and does not colocalize with Ankyrin in vivo. This raises an interesting evolutionary question as to whether a proto-spectrin was bound to a common proto-Ig-CAM and that divergence of the spectrins and Ig-CAMs was accompanied by the acquisition or loss of Ankyrin as an adaptor in the conventional or heavy β-spectrin lineage, or whether the spectrin -- Ig-CAM association came later and this is an example of convergence. Because all of these proteins appear to have emerged at around the time that multicellular animals evolved, this cannot be readily answered at present. Moreover, the separation of conventional and heavy β-spectrins occurred during a period of dynamic concerted evolution that might have erased the evidence within the spectrins themselves. It will be interesting to see if the βH-Ig-CAM association extends to other members of this subfamily such as Hibris, the Roughest ligand (Lee, 2010).


PROTEIN STRUCTURE

Amino Acids - 4097 (Thomas, 1997)

Structural Domains

The published sequence of ßHeavy-spectrin (Dubreuil, 1990), now termed Karst, is partial and represents about 5 kb of a mature 13 kb mRNA. The conceptual translation of the open reading frame in this sequence predicts a peptide with an Mr = 189 x103 which has been shown to be part of a larger protein with an Mr = 430 x103. The sequence of this clone contains a consensus actin binding domain followed by twelve and a half copies of a typical 106 amino acid repetitive domain, and as such, is representative of an N-terminal fragment of a member of the spectrin/a-actinin/dystrophin gene superfamily of actin cross-linking proteins. The sequences of the C-terminal region of dystrophins are extraordinarily conserved and could, in principle, be used to determine the precise relationship of ßH and dystrophin. Another partial cDNA has been recovered that represents the final, approx. 600 amino acids of the ßH protein. There is no detectable similarity to dystrophin in this C-terminal clone, beyond that expected for a member of the a-actinin/spectrin/dystrophin superfamily. Thus Karst is classified as a spectrin and not a dystrophin (Thomas, 1994).

ßHeavy-spectrin is distinguished from the other ß spectrin forms because it contains an SH3 domain; in addition, the PH domains in the C-terminus are unique to ßHeavy-spectrin. Whereas ß-spectrin contains 16 spectrin repeats, ßHeavy-spectrin contains 29 repeats. A major feature found in conventional ß-spectrins is the ankyrin binding site, absent from ßHeavy-spectrin. The ßHeavy-spectrin and ß-spectrin appear to be colinear over the initial 17 spectrin repeats, with an additional conserved 12 spectrin repeats present in the C-terminal half of ßHeavy-spectrin. The unique (nonrepeat) features present in the ßHeavy-spectrin sequence are as follows:

Analysis of the amino acid sequences of alpha-actinin, spectrins, and dystrophin proteins has suggested that all three protein families arose from a single common ancestral protein that was alpha-actinin-like (Dubreuil, 1991). Specifically, alpha-actinin has an N-terminus resembling that of ß-spectrins and dystrophins, a short repeat alpha-helical motif common to the whole family, and Ca2+-binding EF-hands at the C-terminus related to those of alpha-spectrins and dystrophins. The spectrin repeats are reiterated a distinct number of times in each protein, resulting in a characteristic actin-crosslinking distance: alpha-Actinin has 4 repeats; ß-spectrin 17; alpha-spectrin 20; dystrophin 24, and ßHeavy-spectrin 30 (Thomas, 1997)


karst: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 August 2016

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